HEPATOLOGY HIGHLIGHTS *
Authors: Harvey J. Alter
Journal: Hepatology (2005)

Zhang and coworkers have dissected the intracellular events wherein endogenous interferon (IFN) protects hepatocytes from viral damage but also appears to be overcome by the inhibitory effects of hepatitis C virus (HCV)-associated proteins. Rapid induction of type I IFNs (IFN α/β) is a central event in initiating the innate antiviral response. This is a complex process, sequentially resulting in activation of NF-kB and IFN regulatory factor-3 (IRF-3), transcription factors that trigger the release of IFNβ and IFNα1, activation of IFN-stimulated gene factor 3 complex, and upregulation of IRF-7. Ultimately, interferon-stimulated genes generate proteins that impair viral gene expression and replication. Zhang et al. now examine the reciprocal IFN-HCV interactions within hepatocytes. They first demonstrated that unstimulated human primary hepatocytes, and the liver cell lines Huh 7 and Hep G2, express relatively high levels of intracellular IFN-α and that this spontaneous IFN-α production was significantly suppressed in HCV replicon cells. Having shown that HCV suppresses intracellular IFN-α, they examined the reciprocal event, IFN-α inhibition of HCV replicon expression. They showed that HCV replication was enhanced when intracellular IFN-α expression was inhibited by IFN-α siRNA or functionally blocked by antibodies directed at the IFN-α receptor. Conversely, when intracellular IFN-α was induced by IRF-7, there was a corresponding decrease of HCV RNA in replicon cells. In studying the underlying mechanism of HCV-mediated suppression of IFN-α production, they demonstrate that the primary effect is the ability of HCV nonstructural proteins, particularly NS5A, to inhibit the actions of IRF-7. These and similar observations by Foy et al. on NS-3 inhibition of IRF-3 are very important additions to our understanding of how HCV escapes the early intracellular innate immune response and thus gains a foothold that fosters viral persistence. Perhaps the replication efficiency of any given viral strain is dependent on the extent to which it can suppress the endogenous and virus-activated intracellular immune response. When viral suppression of this response is strong, persistence may be inevitable. Speculatively, the difficulty in replicating HCV in culture may be due to this strong constitutive intracellular IFN production. Perhaps, the recently described fulminant HCV strain, that can for the first time be grown in culture, is uniquely adapted to maximally suppress innate immunity and thus replicate unopposed in hepatocytes. Even more speculatively, maybe the fulminant hepatitis itself was a manifestation of this high replicative capacity in a massive number of liver cells rendered defenseless by uniquely inhibitory HCV nonstructural proteins. (See HEPATOLOGY 2005;42:819-827.) The underlying mechanisms of persistent HCV infection remain enigmatic despite numerous studies of viral survival strategies and host innate and adaptive immune responses. One of the most consistent findings is that both CD4+ and CD8+ responses are impaired in those with chronic infection compared to those who spontaneously recover. However, questions remain as to whether this impairment is the cause or the consequence of persistent HCV infection and whether measurements of peripheral T cells fully reflect cell-mediated immune responses in the liver. Spangenberg and coworkers address these issues by isolating intrahepatic T cells from 27 HLA-A2–positive patients and studying them by tetramer assays, intracellular cytokine staining, ELISPOT assays, and antigen-specific cell proliferation. Their important findings can be summarized as follows: (1) intrahepatic HCV-specific CD8+ T-cell responses were detectable in 17 of 27 patients (63%) and targeted an average of 1.3 of the 4 HLA-A2–restricted epitopes tested, primarily in nonstructural coding regions; (2) although a comparable number of patients (59%) had HCV-specific CD8 reactivity in the peripheral blood (PB), the frequency of these cells was significantly lower in PB than in the liver; (3) importantly, only a minority of intrahepatic tetramer+ HCV-specific T cells secreted IFN-γ after stimulation with the cognate peptide, suggesting that a large proportion of intrahepatic HCV-specific CD8 cells are functionally impaired (see Fig.); this was also true in PB. In contrast, Flu-specific CD8 cells produced IFN-γ in the vast majority of patients; (4) intrahepatic HCV-specific CD4+ T-cell responses were observed in 8 of 10 patients tested, but these responses were generally weak; (5) failure to clear virus was not due to the induction of T-cell escape mutants. In sum, this study shows that HCV specific CD8+ T cells do traffic to the liver, but that the virus can continue to proliferate despite their presence. As shown in earlier studies of PB, HCV-specific CD8+ cells appear to be dysfunctional, particularly in their ability to secrete IFN-γ. Once must then consider the basis of this dysfunctionality. One possibility is the lack of appropriate CD4+ T-cell help. Clearly, many studies have shown impaired CD4+ T-cell proliferative and functional responses in the PB of patients with chronic HCV infection and this study demonstrates similar CD4 deficits within the liver. This is basically a dysfunctional T-cell family. There are many other possible explanations for failure to clear HCV in the majority of patients, and these include induction of regulatory T cells, T-cell tolerance induced by high viral antigen levels and specific suppression of T-cell activation by viral proteins, particularly NS-3 and NS-4. Undoubtedly, the HCV-specific immune defect will prove to be multifactorial. This study has embellished our knowledge of HCV survival at the hepatocyte level and thankfully also shown that measurements in the PB are fairly well reflective of the intrahepatic state, thus adding substance to conclusions reached in earlier studies of PBMCs. This study of chronically infected patients could not address whether the observed T-cell defects are the cause or consequence of persistent infection, but similar studies during acute hepatitis C suggest that these impairments are present very early in infection and thus more likely causative then reactionary. Studies of the HCV-specific immune response have become increasingly sophisticated and respected. HCV immunologists can no longer say, “what are we, chopped liver?” (See HEPATOLOGY 2005;42:828-837.) The liver field is scarred with algorithms purporting to predict fibrosis/cirrhosis. Several have been previously highlighted in this column, and I would not address them again except that the “Lok Model,” encompassing platelet count, AST/ALT ratio and INR, has now been analyzed in the context of the HALT-C trial and the large number of subjects (1,141) allows for a comprehensive analysis of this simple model and also opens the door to a general discussion of the clinical value of any such models. The HALT-C trial by its very nature selected patients with severe fibrosis and thus did not encompass the full spectrum of patients with chronic hepatitis C. Hence, of the enrolled subjects, 429 (38%) had cirrhosis and 712 (62%) had Ishak stage 3-4 fibrosis. Using logistic regression models and ROC curve analysis, a regression formula using platelet count, INR and AST/ALT ratio was judged optimal and tested in a training set and a validation set. The key findings were that a cutoff value of <0.2 to exclude cirrhosis misclassified 7.8% of patients with cirrhosis (NPV= 86%) and a cutoff of >0.45 to identify cirrhosis misclassified 14.8% (PPV=75%). Importantly, 48.5% fell between the two cutoff values and could not be classified accurately. Although the tests used are routine, the predictive formula is complex and thus the investigators developed a convenience model that could be used without need for sophisticated calculations. It was shown that in patients with a platelet count of <100,000, an AST/ALT ratio >1 and an INR >1, the predicted probability of cirrhosis was 73% and the observed prevalence of cirrhosis was 79%. In patients with a platelet count >200,000, AST/ALT <1 and INR <1, the predicted probability of cirrhosis was 19% and the observed prevalence 12%. Overall, this predictive formula using routine assays works reasonably well, but how valuable are such algorithms in providing information that would negate the need for liver biopsy and provide the basis for accurate treatment decisions? My enthusiasm is curbed by the fact that 50% of patients have values that cannot be interpreted and that the PPV is only 75% in the remaining half. My main concern is that such algorithms distinguish cirrhosis from noncirrhosis, but what is needed most is a formula that can distinguish the absence of fibrosis or stage 1 fibrosis from F2, F3, or F4 fibrosis, stages where critical treatment decisions have to be made. It is in making these distinctions that the liver biopsy is most helpful, although there is some inherent procedural risk and some inaccuracy due to sampling error. It would seem that a non-invasive marker that could fine tune the stage of fibrosis and avoid the need for biopsy would have to be specific for stellate cell activation or collagen matrix formation or liver stiffness (see next section) and not simply based on markers of inflammation, portal hypertension, or impaired liver synthetic function. The latter are markers of late events in the pathological process and their clinical utility is limited thereby. The perfect predictive model is not yet available, but one prediction that cannot fail is that we will see more predictive models in the future. Fortunately, “most of our future lies ahead.” (See HEPATOLOGY 2005;42:282-292.) On the same theme, Colletta et al. compared the biochemical algorithm, FibroTest, with the shear elasticity probe, Fibroscan, in 40 untreated HCV-infected patients with persistently normal ALT who had undergone two liver biopsies. In identifying patients with significant fibrosis, inter-rater agreement was excellent for Fibroscan (weighted kappa = 1) and poor for FibroTest (weighted kappa= −0.041) (see Fig.). At the other end of the spectrum, 26 of 26 patients with minimal fibrosis could have been spared biopsy by Fibroscan interpretation, whereas only 38% would have avoided biopsy using FibroTest. Further, 14 of 14 of patients eligible for treatment (fibrosis score ≥F2) would have been identified by Fibroscan compared with 9 of 14 (64%) by Fibrotest. As indicated above, predictors of fibrosis that are based directly on collagen matrix formation or the degree of tissue resistance seem inherently more useful than predictors that measure the correlates of portal hypertension and hepatitic dysfunction. The former can correlate with fibrous deposition as it evolves from mild to more serious, while the latter will always be dependent on distinguishing very severe scarring from all other histological possibilities. Aside from the striking correlations between biopsy and Fibroscan, the Colletta study shows that 35% of patients with persistently normal ALT had significant progression of fibrosis on biopsy over a median follow-up period of 7.5 years. None of these progressed to cirrhosis and hence would have been missed by algorithms based on hematological and biochemical indices, but the majority was detected by Fibroscan. This rate of significant progression of fibrosis in persons with normal ALT seems quite high. It correlated significantly in this study with a history of excess alcohol consumption and with high viral load. One implication of this study is that HCV-infected patients have to be monitored for progression of fibrosis even in the face of persistently normal ALT. Such monitoring would be especially important in those with past or current alcohol excess and perhaps in those with high viral load, though the latter is controversial. Since the ALT in such patients is by definition an unreliable index of progression of fibrosis one would need to consider liver biopsy or Fibroscan assessment in the follow-up and management of persons with normal ALT, especially since current therapies appear to be equally effective in this subset of HCV-infected individuals. Measures of liver elasticity are a promising innovation—and that's no stretch. (See HEPATOLOGY 2005;42:838-845.) HCV infection has been associated with several autoimmune phenomena, including arthritis, glomerulonephritis, lichen planus, mixed cryoglobulinemia, and circulating autoantibodies, particularly RF. Although anti-LKM1 autoantibodies are the hallmark of autoimmune hepatitis, 5% to 10% of HCV-infected patients also harbor these antibodies. LKM1 antibodies are specifically directed against cytochrome P450IID6 (CYP2D6), a protein located on the cytoplasmic side of the endoplasmic reticulum of hepatocytes. Marceau and coworkers intensively studied the sera of 3 HCV+ patients with LKM1 antibodies. They purified and concentrated the antibodies and then used them as the selecting matrix with a phage display library. Retained phages eluted with CYP2D6 fusion protein were purified and sequenced. The sequences obtained were compared to each other and to GeneBank sequences of CYP2D6 and HCV-1a. Nineteen mimotopes obtained by phage display were then used to find statistically significant pairs (SSPs) for the identification of conformational epitopes believed to be the target epitopes in HCV infection compared to the linear epitopes that characterize LKM1 reactions in autoimmune hepatitis. Mimotope sequences were compared with HCV protein sequences and core, E2, NS3, and NS5a were found to have homologies with several mimotopes. Three SSPs were identified and mapped on the CYP2D6 crystallographic model, where it was shown that amino acids 254-288 of CYP2D6 contain a putative conformational epitope mimicked by the mimotopes identified by HCV+/LKM1+ sera. Their premise then was that if anti-LKM1 sera purified for CYP2D6 binding could immunoprecipitate an HCV protein this would indicate that the same antibody in HCV+/LKM1+ sera binds to both CYP2D6 and to an HCV protein. It was demonstrated that each of the sera immunoprecipitated NS3, NS5a, or both, indicating that molecular mimicry and cross-reactivity at the B-cell level exists between CYP2D6 and NS3 and NS5a proteins from HCV. The regions of NS3 and NS5a that cross-react with CYP2D6 are highly conserved among HCV genotypes. From this complex analysis of 3 patients it appears that Marceau et al. have uncovered the reason for anti-LKM1 antibodies in patients with hepatitis C. The key finding in this study is that antibodies against Ns3 and NS5a can cross-react with CYP2D6 and hence that initial reactivity to a viral antigen can lead to reactivity against self-antigens. In a larger context, similar reactions may explain many autoimmune diseases wherein the associated virus has not as yet been identified. Thus, this molecular approach to elucidating the mechanism of LKM1 antibodies in HCV infection may ultimately have broader implications for autoimmune diseases in general. (See HEPATOLOGY 2005;42:675-682.)